This application for a Mentored Clinician Scientist Development Award (K08) seeks support for Michelle Pietzak, M.D., who has recently completed her fellowship in Pediatric Gastroenterology and Nutrition and joined the faculty as an Instructor of Pediatrics at Childrens Hospital Los Angeles (Assistant Professor pending). Under the mentorship of Kwang Sik Kim M.D., Dr. Pietzak will continue to pursue her basic investigations into the mechanisms by which Escherichia coli (E. coli) is able to translocate across intestinal epithelium and cause sepsis. E. coli is a leading cause of severe bacterial infections in premature infants, neonates, immunocompromised hosts, and children with central lines and primary intestinal diseases. Dr. Kim is a prominent researcher in the field of the pathogenesis of E. coli meningitis, having (a) established the virulence roles for the K1 capsular polysaccharide, outer membrane protein A, and S fimbriae using in vitro and in vivo models of the blood brain barrier developed in his lab and (b) identified several novel genes thought to be responsible for invasion of E. coli strain RS218 across the blood brain barrier, using these same models. E. coli strain RS218 is a clinical isolate from the cerebrospinal fluid of a human neonate with E. coli meningitis. Dr. Pietzak's project is focused on investigating the mechanisms by which E. coli strain RS218 is able to penetrate the intestinal epithelial barrier, both in vitro and in vivo. In earlier studies, Dr. Pietzak has used strain E44, a spontaneous rifampin resistant mutant of E. coli strain RS218, to demonstrate that this bacterium is able to invade two intestinal epithelial cell lines, Caco-2 and C2BBe-1. The specific aims of this proposal are to further characterize the invasive phenotype of E-44 in vitro, using both gentamicin invasion assays and a trans-well system. An in vivo model, using neonatal rats, will also be used to test the invasive phenotype of E-44. Environmental factors, which mimic the intraluminal gastrointestinal milieu, will be employed to examine their effects on E. coli invasion and translocation in vitro. The virulence of TnphoA transposon mutants, created in our lab and already shown to be noninvasive to the blood brain barrier in vitro and in vivo, will be determined for the intestinal epithelial barrier both in vitro and in vivo. The precise contribution of these genes to the intestinal translocation of E. coli strain RS218 will then be examined using additional molecular techniques. Under Dr. Kim's mentorship, and with Institutional support, Dr. Pietzak will be able to perfect her techniques in cell tissue culture and animal models of bacterial infection, as well as acquire new knowledge and skills in the sciences of bacterial genetics and molecular pathogenesis. Nurturing these ambitions in a collaborative environment will help Dr. Pietzak achieve her goal of becoming an independent investigator, who hopefully will be able to bring the results of her investigations from the benches of the lab to the bedsides of chronically ill children with gastrointestinal and other diseases.